formation of large macropores (>1,200 m) resultingfrom greater earthworm burrowing in the biochar-amended soil.Conclusion We found no evidence to suggest applica-tion of biochar influenced soil porosity by either directpore contribution, creation of accommodation pores, orimproved aggregate stability.

The specific mechanisms by which biochar influ-ences water retention, macro-aggregation, and soil sta-bility are poorly understood (Sohi et al. 2009b). Wepropose that biochar application may influence soilporosity and thus soil water retention via three mecha-nisms (1) direct pore contribution from pores within thebiochar, (2) creation of packing or accommodationpores between biochar and the surrounding soil aggre-gates, and (3) through improved persistence of soil poresdue to increased aggregate stability.

A number of researchers have suggested that due tothe highly porous nature of biochar, its application tosoil may improve soil physical properties through directcontribution of new pores (Atkinson et al. 2010; Downieet al. 2009; Major et al. 2009; Sohi et al. 2010; Verheijenet al. 2010). Despite the apparent link between biocharporosity and soil porosity, surprisingly few studies havereported the pore size distribution of the biochar used forsoil amendment. Biochar pore size is known to varyover several orders of magnitude depending on feed-stock and pyrolysis temperature (Thies and Rillig 2009).Major et al. (2009) suggested that 95 % of pores withinmost biochars are less than 0.002 m diameter; howev-er, biochars have also been shown to contain a largedegree of macroporosity in the 1 to 10 m range(Downie et al. 2009; van Zwieten et al. 2010).Verheijen et al. (2010) proposed that direct pore contri-bution from biochar potentially increased water storagebetween 10,000 and 1,000,000 kPa and thus poten-tially increased the number of pores between 0.03 and0.0003 m diameter in the amended soil. However,most plants are not able to extract soil water from poressmaller than 0.2 m (below the permanent wilting point1,500 kPa) or utilise the transient water passingthrough pores greater than 30 m diameter (above fieldcapacity 10 kPa). Therefore according to Verheijenet al. (2010) whilst biochar amended soils may havehigher total porosity or lower bulk density, the PAWC orwater holding capacity would have remained unchanged.

Few studies have considered that biochar applicationto soil may create accommodation pores between thebiochar particles and the soil aggregates. The size and

proportion of accommodation pores is potentially influ-enced by the size of the soil aggregates, the size of thebiochar particles, and the degree of compaction or set-tling following incorporation. Evidence for the creationof accommodation pores following biochar applicationis limited. Jones et al. (2010) reported that application of40 and 80 Mg ha1 of green waste biochar to bauxiteprocessing residue coarse sand significantly decreasedmacroporosity (pore diameters >29 m) whilst signifi-cantly increased mesoporosity (pore diameters between0.2 and 0.29 m). Increased mesoporosity was attribut-ed to the biochar partly filling large voids between thecoarse sand particles. Evidence from pot trials alsosuggest that short-term changes in pore-size distributionfollowing biochar application may result from aggregatesettling and thus changes to accommodation pores(Eastman 2011; Novak et al. 2012). However, the extentto which biochar application influences resettling ofdisturbed soils has not been specifically investigated.

Currently, there are surprisingly few studies whichdemonstrate that biochar application significantly im-proves the physical properties of agricultural soils.Further research is required to evaluate the potentialfor biochar as an in situ soil amendment. To be relevantto agriculture, these studies need to be conducted in situin agricultural production systems employing biocharscontaining a large proportion of pores within the PAWCpore size range (0.230 m).

The trial was established in November 2009 atMountain River in the Huon Valley, Tasmania (42572.91S, 147552.13E) during replanting of an existingapple orchard. Soils were classified according to Isbell(2002) as a Bleached Mottled Grey Kurosol (texture-contrast) or Planosol (IUSS Working Group WRB2006) developed on Permian Mudstone with a minorcontribution from Jurassic dolerite colluvium. The soilprofile was described and classified according to

The site was levelled and re-mounded 1 week after theremoval of the old trees. The trial design consisted of arandomised complete block with four treatments andfive replicates, trees were blocked on position withinthe tree-row. Each replicate was 3.18 m long and 1 mwide and contained three trees. The four treatments wereuntreated control, biochar, compost, and biochar+com-post. The biochar was sourced from Pacific Pyrolysis,Somersby, NSW (Australia). The feedstock consisted ofacacia whole tree green waste which underwent pyrol-ysis in a continuous flow kiln at temperatures up to550 C for 3040 min. The biochar treatments wereapplied on the 2nd of November 2009, with each repli-cate receiving 15 kg of biochar, equivalent to 5 kg pertree space or 47 Mg ha1. The biochar was spreadevenly by raking across the mound and was incorporat-ed to approximately 10 cm depth. Each block received15 kg of biochar whichwas equivalent to 5 kg per tree or47 Mg ha1. The orchard was replanted with Naga-FuNo 2 Fuji trees on M26 rootstock with a Royal Galainterstem. Tree spacing within the row was 1.06 m, and4.5 m between rows. The compost and mixed biocharcompost treatments were not sampled in this study andthus are not reported. All sampling and measurementswere conducted from the biochar and control treatmentsin replicates 2, 4, and 5 as to avoid disturbing perma-nently installed moisture probes and flux meters inreplicate 1.

Biochar porosity

Six transverse images of a biochar particle were obtain-ed at 300 times magnification using a Hitachi SU-70

where D is the average pore diameter (in nanometre), Vis the total intrusion volume (in millilitre per gram), andA is the total pore surface area (in square metre pergram). Bulk density was calculated from the mass ofbiochar and the total intrusion volume. Apparent densityof the biochar skeleton was calculated from the sum ofthe volume of the solid (non-intruded) material. Porositywas determined as the volume of pores divided by thesample volume. The characteristic length of the poreswas calculated from the Washburn equation from thepressure at which percolation through the porous mediafirst occurred. Tortuosity was calculated as the ratiolength of the path described by the pore space lengthto the length of the shortest path across a porous mass(Webb 2001).

Amount of applied biochar

The mass of biochar was determined from the 560 and249 cm3 cores obtained for determination of drainablemacroporosity and soil water retention by the evapora-tive flux method. Biochar was recovered by dis-aggregating the soil cores in a 30-L container thensieving the floated material to extract the >250 mbiochar fraction. The floated material was dried at105 C for 24 h. Foreign material including roots andparticulate organic matter was manually removed withtweezers before determining the oven-dried mass ofrecovered biochar. The size distribution of the biocharprior to soil incorporation was determined by dry siev-ing in triplicate for 3 min using a stack of 4,000, 2,000,

1,000, 350, 250, and 125 m sieves. The mean weightdiameter of the biochar was 3.84 mm. The averageproportion of biochar less than 250 m was 1.67 %(SD 0.10 %) of the total biochar mass. The effect ofreplicate location and core sample size on biochar masswas investigated by univariate ANOVA in SPSS V 20.

Soil bulk density and porosity

Soil bulk density was determined by the intact coremethod (Cresswell and Hamilton 2002) in May 2012.From each of the three control and biochar-amendedreplicates, three 5080 mm cores, plus three 75100 mm cores, and three 6061 mm cores were obtain-ed from which the bulk density was determined.Gravimetric moisture content was determined by dryingthe entire core at 105 C for 24 h. Total porosity wascalculated from bulk density assuming a particle densityof 2.65 g cm3 and 98 % saturation. The direct effect ofbiochar porosity on soil density was determined bycalculating the soil density without the porosity contrib-uted by the biochar. The volume of biochar was calcu-lated from the biochar density (0.51 g cm3) determinedby mercury porosimetry and the mass of recoveredbiochar in each sample in which the volume and massof recovered biochar were removed from the originaldry soil mass and soil volume. This gave the density ofthe soil as would have occurred without the direct con-tribution of pores from the biochar. The effect of biocharapplication on bulk density and total porosity was in-vestigated by univariate ANOVA in SPSS V 20.

Soil moisture

Soil moisture was measured in triplicate 10 cm from thecentre tree in each replicate every 2 weeks betweenJuly 2010 and April 2013 using an ICT InternationalPty Ltd TDR based Moisture Probe Meter MPM-160-Bwith a 6-cm-long probe. The effect of biochar applica-tion on bimonthly soil moisture was investigated usingunivariate ANOVA in SPSS. Differences in soil mois-ture content between treatments were demonstrated bycalculating the cumulative soil moisture content overtime from the bimonthly soil moisture sampling.

Drainable porosity and field capacity

The drainable porosity was determined by desorptionusing ceramic suction plates at 0.0, 0.1, 1.0, 3.0, and

350 Plant Soil (2014) 376:347361

10.0 kPa (field capacity) according to Cresswell(2002) and Reynolds and Topp (2008). Three replicate10075 mm intact cores were obtained from each of thecontrol and biochar replicates when the soil profile wasmoist but below field capacity. The cores were incre-mentally brought to saturation in a 0.01 M CaCl2 solu-tion over a period of 45 days prior to analysis, and thenpicked to expose open pore faces before being imbed-ded onto the suction table with diatomaceous earth. Thecores were allowed to equilibrate at each matric poten-tial over a period of 512 days until the outflow ceased.The effect of biochar application on drainable porosityand field capacity was determined by univariateANOVA in SPSS V 20.

Soil water retention

The soil water release curve was determined by theevaporative flux method according to the proceduredescribed by Wendroth et al. (1993) and Peters andDurner (2008) using the HYPROP apparatus andtensioVIEW software (UMS 2013). The soil water re-tention curve was fitted using both the van GenuchtenMualem equation (van Genuchten 1980) and the bimod-al vanGenuchtenMualem equation (Durner 1994), withand without the soil moisture content at 1,500 kPawhich was predetermined by the pressure chamber anal-ysis. The lowest RMSE (0.0015) and highest absoluteAkaike Information Criterion (2,399) (Akaike 1974)indicated that the best model fit was achieved for thebimodal van GenuchtenMualem model (Durner 1994)without the supplementary 1,500 kPa data, in which

Se h X2

j1 j 1 j hj j n j 1=n j1

Se r .

s r

where Se is the effective saturation, h is the matricpotential, j is an index of the parameters of each vanGenuchten functions, j is the weight of both partialfunctions, alpha () is an empirical parameter related toair entry, is the soil moisture content, s is the saturatedor field saturated soil volumetric water content, r is theresidual soil volumetric water content, and n is a dimen-sionless empirical constant. As the data pairs wereunique to each soil core, the volumetric soil moisturecontent was calculated from the bimodal vanGenuchtenMualem equation at matric potentials of 0,

10, 20, 30,and 50 kPa within the measurementrange of the evaporative flux approach, and by extrap-olation to 100, 300, 1,000, and 1,500 kPa for eachsoil core. Treatment and plot effects were thus able to beinvestigated by univariate ANOVA for each of the bi-modal van GenuchtenMualem soil parameters and thepredetermined matric potentials.

The PAWC was calculated as the water-filled porespace between field capacity, said to exist at 10 kPaand the permanent wilting point (PWP) at 1,500 kPa(Brady and Weil 2010; James 1988; Marshall andHolmes 1988). The pore size distribution was estimatedfrom the soil water characteristic according to theYoungLaplace equation which assumes that the poresare perfectly cylindrical, uniform, and equally drained.The YoungLaplace equation is approximated by

The PWP was determined by pressure chamber analysisat 1,500 kPa using air-dried

each plot at five supply potentials () of 0.95, 0.55,0.35, 0.15, and 0.05 kPa. The soil moisture prior toinfiltration was determined from five 60 mm60 mmintact cores per replicate according to the proceduredescribed by Cresswell and Hamilton (2002). The un-saturated hydraulic conductivity was calculated accord-ing to the procedure developed by Ankeny et al. (1991)and Reynolds and Elrick (1991), and presented inMcKenzie et al. (2002) in which;

Kx;y Gdx;yqx .

r 1 Gdx;yr

qx.qy

PK Kx;yexp x;y

x;y ln qx

.qy

. x y

P x.

x y

where Kx,y is the average hydraulic conductivity for datapairs, K() is the unsaturated hydraulic conductivity (inmillimetre per hour), x,y is the soil structure parameter,P is a shape parameter, r is the radius of the disk (incentimetre), x,y are the supply potentials (incentimetre), qx,y is the steady state infiltration rate (incubic centimetre per minute), Gd is a shape parameter=0.25, equals (x+y)/2), and x,y represent measure-ments at sequentially less negative supply potentials.The flow weighted mean pore diameter was determinedaccording to Philip (1985)) in which;

FWMPD 7:4 lnK2=K1

21

Here, FWMPD is the flow weighted mean pore di-ameter (in millimetre), and K1 and K2 are the first andsecond hydraulic conductivities (in millimetre per hour)at 1 and 2, where 1 and 2 are the first and secondsupply potentials (in millimetre). Infiltration and unsat-urated hydraulic conductivity data were log transformedprior to analysis by univariate ANOVA in SPSS.

Aggregate stability

Aggregates were sampled from 0 to 3 cm depth usingthree shovel loads per treatment. Aggregates weretransported in open trays to reduce compaction anddeformation. Aggregates were air-dried at ambient tem-peratures for 3 days before being sieved to obtain the12-mm fraction. The 12-mm fraction was oven-driedat 40 C for 24 h to ensure consistent starting moisturefor all treatments. Air-dried 12-mm aggregates wereimmersed in water for 1 min on a 250-m sieve then

where AS12mm>250 m is the aggregate stability of the12-mm fraction measured as the proportion of totalaggregates (minus the resilient stone, root, and biocharfraction) retained on a 250-m sieve (in gram per gram);R>250 m is the oven-dried retained persistent aggregatesand coarse fraction greater than 250 m (in gram);SB>250 m is the oven-dried resilient stone, root, andbiochar component greater than 250 m (in gram); Sairis the air-dried (40 C) mass of aggregates includingresilient fraction prior to sieving; and is the gravimetricmoisture content of the air-dried 12-mm fraction (ingram per gram). The effect of biochar application onaggregate stability was investigated by univariateANOVA in SPSS v20.

Results

Biochar porosity

SEM demonstrated that the average pore size from thesix SEM images of a single biochar particle (measuredalong the longest pore axis) ranged from 0.844 m (sd0.13 m) to 235 m (sd123 m), with the mean poresize ranging from 13.09 m (sd20.03 m) to 7.08 m(sd6.98m) for the six images, depending on pore andmeasurement orientation. Pores were highly ellipticalsuggesting that the original porous structure of the woodfeedstock had become distorted during pyrolysis. Poresize distribution was highly skewed with 95 % of poresbeing less than 14.4379.15 m (range due to poreelongation) (Fig. 1).

Mercury porosimetry revealed that the average min-imum pore diameter was approximately 0.1 m, inwhich 95 % of all pores were less than 22 m diameter.

352 Plant Soil (2014) 376:347361

The average median pore diameter can be considered torange from 0.413 m. The characteristic length ofpores averaged 44 m which was also reflected in theaverage tourtuosity value of 6.6 indicating pores were4.28.0 longer than they were wide (Table 1).Differences in biochar porosity determined by SEMversus the mercury porosimetry were considered minor,and largely due to detection of asymmetrical pore prop-erties by SEM.

Biochar recovery

The mean biochar recovery was 3.39 g 100 cm3 (SD1.46 g 100 cm3); however, themass of recovered biochar(>250 m) varied between individual samples from 1.06to 6.75 g 100 cm3. Differences in amount of recoveredbiochar between replicates and the two soil sample vol-umes were not significant. Analysis of the 100-mm-diameter soil cores demonstrated that the volume of therecovered biochar was 6.53 % of the total soil volume.

Soil bulk density, total porosity, and saturated watercontent

Biochar application significantly reduced the soil bulkdensity in all replicates and for all three core samplesizes (F=59.226, P=0.015) (Fig. 3a). Consequently,biochar amendment resulted in significantly higher total

The lower bulk density of the biochar-amended soildid not result from direct pore contribution from thebiochar itself, as the bulk density of the biochar excludedtreatment (effects of biochar porosity had been removedfrom the soil volume) was significantly lower than theunamended control (F=320.26, P=0.0001) (Fig. 3a).

Soil moisture

Biochar application had no significant effect on soilmoisture content (Fig. 4a) or cumulative soil moisturebetween July 2010 and May 2013 (Fig. 4b).

Drainable porosity and field capacity

Biochar application had no significant effect on thedrainable porosity between 1.0 and 10 kPa, nor on

the field capacity at 10 kPa. However, biochar appli-cation significantly increased the saturated soil moisturecontent (F=132.878, P=0.007), and soil moisture con-tent at 0.1 kPa (F=32.639, P=0.029). This supportsthe previous finding that application of biochar signifi-cantly reduced the soil bulk density and increased thetotal porosity, presumably due to the creation or preser-vation of large pores (>300 m) in the surrounding soil(Fig. 5).

Soil-water retention and plant available water

Determination of the soil-water retention function by theevaporative flux method revealed considerable variabil-ity within treatments and between replicates. Biocharapplication was found to have no significant effect on(1) the bimodal van GenuchtenMualem soil waterparameters (1,2, s, r, n1,2, ), (2) the measured equil-ibration potentials between 10 and 50 kPa, (3) theextrapolated equilibration potentials between 100 and

in the formation of macropores at least 1,200 m diam-eter, but not smaller than 660 m diameter. This findingcontrasts to other studies conducted on in situ agricul-tural soils which have reported that biochar applicationhad no significant effect on saturated hydraulic conduc-tivity (Eastman 2011; Major et al. 2012). The formationof these large macropores was attributed to a fourth andpreviously unreported mechanism by which biocharmay influence soil porosity: that is, increased inverte-brate burrowing. At the time of sampling, earthwormnumbers were visibly higher in the biochar-amendedsoil than the untreated control. Consequently, the in-creased number of large macropores (>1,200 m), andthus increased total porosity, saturated water content andnear saturated hydraulic conductivity (0.25 kPa) ofthe biochar-amended soil was attributed to increasedearthworm burrowing. Few studies have investigatedthe effect of biochar application on invertebrates(Lehmann et al. 2011). In their review, Weyers andSpokas (2011) concluded that biochar may have short-term negative impacts on earthworm population densityand total biomass. However, there was little evidence tosuggest biochar had any long-term effects on earthwormdensity or total biomass. In a behavioural experiment,van Zwieten et al. (2010) showed that earthworms pre-ferred a biochar-amended Ferrosol but had no preferencefor biochar in a Calcarosol. Gomez-Eyles et al. (2011)however found that biochar application in a contaminatedsoils resulted in the loss of earthworm mass and condi-tion, whilst Busch et al. (2012) demonstrated biochar hadno effect on earthworm avoidance in a contaminated soil.Earthworm response to biochar application appears todepend on biochar type, soil type, and time.

Results demonstrate that substantial within- andbetween-replicate variation existed in the amount ofrecovered biochar, and physical soil properties such asbulk density, hydraulic conductivity, soil water reten-tion, and aggregate stability. Although variation in theamount of recovered biochar (1.066.75 g 100 cm3)may have influenced measured values, this effect wasonly apparent for bulk density (Fig. 2b). Furthermore,data indicates that soil hydraulic properties such asdrainable porosity, hydraulic conductivity, and the soilwater retention function varied due to the high spatialvariation in soil pore size and pore arrangement at thesite, not biochar amendment. Orchard soils are expectedto have a higher degree of pore space variation thanarable soils as they are relatively undisturbed, allowingdevelopment of soil structure by processes such as

freezethawing, bioturbation, microbiological activity,and shrink-swelling (Hillel 1998). Consequently, in con-trast to pot trials in which natural soil structure isdestroyed and homogenised, in situ studies of orchardsoils require treatment effects to be substantially greaterthan pot trials in order to yield a statistically significantchange in soil physical properties. In this study, appli-cation of 47 Mg ha1, acacia biochar had no significanteffect on a range of soil hydraulic properties due in partto the high natural variation in soil physical properties.Consequently in order to produce a statistically signifi-cant effect, biochar would need to be applied at rates inexcess of 50 Mg ha1, which is both physically andeconomically prohibitive in commercial orchards.

Conclusion

We proposed three mechanisms by which biochar ap-plication might increase soil porosity. They were (1)direct pore contribution from the pores within the bio-char, (2) creation of packing or accommodation pores,and (3) improved aggregate stability. Mercuryporosimetry and SEM analysis demonstrated that theacacia biochar used in this study contained pores be-tween approximately 0.1 and 240 m in diameter, with95 % of all pores being less than 22 m. Consequently,application of the acacia biochar was expected to in-crease plant available water through direct pore contri-bution by increasing the proportion of pores betweenfield capacity (30 m) and the permanent wilting point(0.2 m), and to a lesser extent macroporosity (porediameters >75 m). However, application of47 Mg ha1 of acacia green waste biochar had nosignificant effect on the following: drainable porosity(1.0 and 10 kPa), field capacity, PAWC, PWP, the vanGenuchten soil water retention parameters (1,2, s, r,n1,2, ), or soil moisture content. We found no evidenceto suggest that biochar application directly influencedsoil porosity through either direct pore contribution, thecreation of accommodation pores, or increased aggre-gate stability as we had speculated. However, thebiochar-amended soil had significantly higher near-saturated hydraulic conductivity (0.25 and0.10 kPa), total porosity, and soil water retention at0.1 kPa resulting from the presence of largemacropores (> 1,200 m). These large macroporeswere attributed to increased earthworm burrowing,based on unrecorded observations of earthworm

358 Plant Soil (2014) 376:347361

presence during the experiments. More research is re-quired to verify this hypothesis.

Our study demonstrated that despite use of a biochardominated by pores within the PAWC range, applicationat 47 Mg ha1 to a loamy sand soil within an appleproduction system had no significant effect on soil wateravailability or soil moisture content. Lack of a significantdifference in all soil physical properties between the con-trol and biochar treatments resulted in part from the highspatial variation in pore size and architecture at the site.

Acknowledgments This project was conducted as part of thenational apple and pear industry Productivity Irrigation Pests andSoils flagship program and was funded by Horticulture AustraliaLimited using the apple and pear industry levy, voluntary contri-bution from the New Zealand Institute for Plant and Food Re-search, and matched funds from the Australian Government. Wethank Justin Direen for assistance with trial establishment andBenedicte Patin, Steve Patterson, Jocelyn Parry-Jones, and AnnaWrobel-Tobiszewska for assistance with field work. Assistancewith SEM and mercury porosimetry was gratefully received fromDario Arrua and Jocelyn Parry-Jones. Thanks to Drs CarolineMohammed and Alieta Eyles for valuable comments on an earlierdraft of the manuscript. This work was conducted whilst the firstauthor was seconded from the Department of Primary Industries,Parks, Water and Environment.

References

Akaike H (1974) A new look at statistical model identification.IEEE Transactions on Automatic Control AC-19:716723

Shackley S, Sohi SP (2010) An assessment of the benefits andissues associated with the application of biochar to soil.Department for Environment Food and Rural Affairs andDepartment of Energy and Climate Change

ResultsBiochar porosityBiochar recoverySoil bulk density, total porosity, and saturated water contentSoil moistureDrainable porosity and field capacitySoil-water retention and plant available waterInfiltration and hydraulic conductivityMicroporosity and the permanent wilting pointAggregate stability